Chemiluminescent compounds are those which undergo a chemical reaction resulting in the emission of light energy, referred to as luminescence. In such reactions, the product of the chemical reaction is in an electronically excited state capable of undergoing a radiative transition to a ground state with the accompanying emission of radiant energy. The chemiluminescence of dioxetanes when enzyme-triggered is useful in, for example, the field of immunoassay. If the reaction is carried out in solution, the light emission lasts approximately 1-2 minutes. If the reaction is carried out in conjunction with a sample affixed to a membrane or other solid support, the dioxetane intermediate responsible for the light emission has increased stability, resulting in a light emission lasting as much as several days.
Chemiluminescent signals via enzymatic triggering of certain substituted 1,2 dioxetanes have been reported by several groups. In general, such references teach the generation and use of chemiluminescent signals produced by enzymatic cleavage of a labile group from a substituted 1,2-dioxetane. Following the removal of the enzyme-cleavable group, an intermediate anion is formed, which subsequently decomposes by rupture of the dioxetane ring. At least one of the resulting carbonyl compounds formed is a light-emitting fluorophore.
U.S. Pat. No. 4,857,652 to Schapp discloses light producing 1,2-dioxetanes of the formula ##STR1## wherein ArOX is an aryl ring substituted with an X oxy group and A are passive organic groups which allow the 1,2-dioxetane to produce light when triggered by removing X. X is a chemically labile group which is removed by an activating agent. The 1,2-dioxetane compounds can be triggered to produce light at room temperatures.
U.S. Pat. No. 4,952,707 to Edwards et al., affords a general description of enzymatically-cleavable 1,2-dioxetanes. This patent describes enzymatically-cleavable chemiluminescent 1,2-dioxetanes having the formula: ##STR2## wherein R.sub.1 is hydrogen, or a bond when R.sub.2 is a substituent bound to the dioxetane ring through a spiro linkage, or an organic substituent that does not interfere with the production of light; R.sub.2 is a fused polycyclic ring-containing fluorophore moiety having an enzymatically-cleavable, labile ring substituent; and T is a stabilizing group that prevents the dioxetane compound from decomposing before the enzymatically-cleavable labile ring substituent's bond is cleaved.
U.S. Pat. No. 4,956,477 to Bronstein et al., also describes the synthesis of enzyme-cleavable 1,2-dioxetanes, useful for chemiluminescent immunoassays, DNA probe assays, and direct assays for an enzyme.
U.S. Pat. No. 4,959,182 to Schaap describes a method and composition for providing enhanced chemiluminescence from 1,2-dioxetanes. In this method an enzyme cleavable 1,2-dioxetane is mixed with a surfactant and a fluorescent compound attached to a hydrocarbon to form a co-surfactant in a micelle or other structure. This method provides an enhancement of 500 fold in signal for enzyme-triggered chemiluminescence of 1,2-dioxetanes in solution. Moreover, Bronstein et al., (J. Biolumin. Chemilumin. 4, 99-111, 1989) report that bovine serum albumin and other water-soluble macromolecules provide a significant enhancement of chemiluminescent signal generated from enzyme-cleavable 1,2-dioxetanes in solution. All of these enhancers are believed to increase the stability of the anion intermediate and the light-emitting species by keeping them in a hydrophobic environment. However, on a hydrophobic support such as a nylon membrane, the support provides a hydrophobic environment for the anionic species. Consequently, no significant enhancement is provided by these enhancers when the enzyme is immobilized on a hydrophobic support.
AU-A36340/89 to Okada et al. describes a method for enhancing the chemiluminescent signal from enzyme-triggered 1,2-dioxetanes. The enzymatic reaction is performed at the optimum pH for the enzyme. Afterwards the pH is increased by the addition of strong base to enhance the luminescent reaction. Increases in signal from 7 to 59 fold were reported for assays done on polystyrene beads. This method does not produce as large an enhancement as our heat-drying during detection method and requires the use of a caustic chemical, 1 N NaOH.
W089/06650 to Bronstein et al. discusses dioxetanes for use in assays, and including a fluorescent chromophore spiro-bound at the 4-carbon of the dioxetane. The dioxetane has the formula: ##STR3## where X is CR.sub.6 R.sub.8, O, S, or R--R (where each R.sub.7, R.sub.8, and R, independently, is H, alkyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, cycloheteroalkyl, aralkyl, aklaryl, or an enzyme cleavable group). Each R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6, independently, is H, an electron withdrawing group, an electron donating group, heteroaryl, or an enzyme cleavable group, or groups R.sub.1 -R.sub.6 together form a ring. T is a substituted or unsubstituted aryl, polyaryl, cycloalkylidene or polycycloaklylidene group spiro-bound at the 3-carbon of the dioxetane. These dioxetanes are used in an assay to detect a member of a specific binding pair or an enzyme.
U.S. Pat. No. 4,705,847 to Hummelen et al. relates to a process for preparing substituted polycyclo-alkylidene polycyclo-alkanes, such as substituted adamantylidene adamantanes, and the corresponding epidioxy compounds. The polycycloalkylidene polycyclo-alkanes are halogenated, and thereafter the halogenation product is optionally subjected to a substitution reaction. The resulting products are converted to the corresponding epidioxy compounds. Various epodioxy compounds are disclosed which contain a dioxetane ring. These compounds are useful as thermochemiluminescent labels.
The use of 1,2-dioxetanes as labels for thermochemiluminescent immunoassays is reviewed by J. C. Hummelen et al. in Methods in Enzymology, Vol. 133, pp. 531-557, 1986. When heated, the 1,2-dioxetanes decompose thermally into two carbonyl fragments. A fraction of these fragments are formed in an electronically excited state and emit radiant energy upon transition to the ground state. In a thermochemiluminescent binding assay, one binding partner is labeled with the 1,2-dioxetane. After the binding reaction between the labeled partner and the immobilized partner, the support is heated to 150.degree. C. to 250.degree. C. to generate the chemiluminescent signal.
The main distinction between this approach and the instant invention is the use of enzyme-triggered 1,2-dioxetanes. The enzyme is used as the label, and the dioxetane is the substrate. The intermediate that is formed by the enzymatic reaction is thermally triggered to produce enhanced chemiluminescence. Basically, the present approach is a combination of enzyme generated chemiluminescence and thermochemiluminescence. There are two major advantages to the present approach. The sensitivity is greater because of enzyme amplification, i.e., the enzymatic reaction can be allowed to proceed until sufficient intermediate is formed. The second advantage is that lower temperatures are used because the intermediate is not as stable as the thermochemiluminescent dioxetanes used as labels. High stability is not required for the intermediate, as it is in the case of thermochemiluminescent labels, because the intermediate is formed in situ by the enzymatic reaction.
U.S. Pat. No. 4,948,975 to Erwin et al., describes a quantitative luminescence imaging system which provides a means to measure low light levels from luminescent reactions in electromagnetic fields, e.g., microwave radiation, and its use in the areas of chemiluminescent assays and thermal microdosimetry. The effect of the microwave radiation on chemiluminescence described in the patent and in a publication (Kiel, Bioelectromagnetics 4,193-204, 1983) is significantly different from the instant description. The system described involves enzymatic reactions, specifically the oxidation of luminol catalyzed by peroxidase enzymes, in protein gels which are kept wet with solution. The enhancement affected by microwave radiation is due to an increased mobility of substrate (hydrogen peroxide) within the gel, not to an actual enhancement of the chemiluminescent process as presently disclosed.
These and other references broadly describe chemiluminescent processes involving dioxetanes. However, none of the references describe a method to enhance the chemiluminescent signal of enzyme triggered 1,2-dioxetanes on solid supports using drying or a combination of drying and heating the support.
Therefore, it is an object of the present invention to provide a process for enhancing the chemiluminescent signal produced by enzyme-triggered 1,2-dioxetanes. It is a further object of this invention to enhance signal intensity in solid phase assays over a reduced time period and without labor intensive procedures or equipment. A feature of the present invention is that the chemiluminescent signals of the enzyme-triggered 1,2-dioxetanes are increased 4-6 fold employing the drying techniques disclosed herein (and fully 100 fold when the heating step disclosed herein is additionally utilized), over signal strengths detected using conventional techniques in which the membrane is kept wet with substrate solution. An advantage of the present process is its versatility in several applications including solid phase assays such as enzyme linked immunosorbent assays (ELISAs) and DNA probe assays. Further the enhancement is useful for imaging nucleic acid or protein blots where a camera apparatus is used; the present procedure features the capability to capture images quickly and at great intensity, avoiding concerns such as camera cooling and background noise levels.
These and other objects, features, and advantages of the present invention will become more readily appreciated and understood upon having reference to the following description of the invention herein.